The present invention provides high-level, direct recombinant expression of subunit analogs of S1, S2, S3, S4, and S5 of Bordetella exotoxin in E. coli without resort to fusions with portions of heterologous proteins. More particularly, genetically-engineered modifications of the subunits provide a class of Bordetella toxin analogs having the capability to elicit toxin-neutralizing levels of antibodies, and to be substantially free of reactogenic components. Genetically-engineered subunits can be used to produce subunit vaccine(s) which have immunogenic efficacy and are substantially free of reactogenic components.
The term Bordetella exotoxin denotes a group of toxins encoded by the genomes of various species of Bordetella, such as B. pertussis, B. parapertussis and B. bronchiseptica. Other terms commonly used to designate Bordetella exotoxin are pertussis toxin (“PTX”), lymphocytosis-promoting factor (“LPF”), and islet-activating protein (“IAP”).
Whooping cough remains a major cause of infant morbidity and mortality in many parts of the world. Whole-cell Bordetella pertussis vaccines have provided an effective means for controlling this disease. However, the use of such vaccines has been directly correlated with mild side effects and temporally related to more severe, and occasionally fatal, neurological events.
Extensive efforts have been expended in an effort to eliminate the harmful side-effects known to be associated with the current vaccines. These have resulted in the production and testing of acellular vaccines, and in basic research in an effort to develop safer recombinant products. A critical first step toward cloning and developing a recombinant DNA-derived vaccine was sequencing of the pertussis toxin operon and subsequent deduction of the amino acid sequences of the individual subunits. (Locht, C. and Keith, J. M., 1986, Science 232: 1258–1264; Locht et al., 1986, Nucl. Acids Res. 14: 3251–3261; and Nicosia et al., 1986, Proc. Natl. Acad. Sci. USA 83: 4631–4635).
Nicosia et al. (1987, Infect. Immun., 55: 963–967) demonstrated that mRNA encoding each of subunits S1, S2, S3, S4, and S5 of Bordetella pertussis could be efficiently transcribed from the cloned genes in E. coli. Although they purported to show high levels of transcription of the native pertussis toxin polycistronic message, the amount of proteins produced by direct expression was very low or undetectable. Further, fusion proteins which were subsequently synthesized were incapable of eliciting any neutralizing or protective immune responses.
Barbieri et al. (1987, Infect. Immun., 55: 1321–1323) demonstrated the expression of the S1 subunit as a fusion protein in E. coli. This fusion protein contains the first six amino acids of beta-galactosidase, five amino acids encoded by the pUC18 polylinker, followed by amino acids 2 through 235 of the S1 subunit. The S1 fusion protein, produced in low amounts, had only about 25% of ADP-ribosyltranferase activity of authentic or native pertussis toxin.
Locht et al. (Abstract, Modern Approaches to New Vaccines, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., Sep. 9–14, 1986) were able to express a fusion protein containing amino acids 2 through 187 of the S1 subunit. They predicted that the construct would not have toxic activity because they believed it lacked the NAD-binding site associated with the ADP-ribosyltranferase, the enzymatic activity believed to be responsible for the reactogenicity of the toxin. Subsequent experiments with this molecule indicated that this truncated species possessed essentially undiminished enzymatic activity. None of the known prior art subunits or subunit analogs have the capability of eliciting toxin-neutralizing levels of antibodies and are substantially free of enzymatic activity associated with reactogenicity.